DESCRIPTION (Adapted from the application) The overall objective of this application is to gain insight into gene expression in prostate disease. Research in the last project period revealed an oxidative 3alpha-hydroxysteroid dehydrogenase that converts 5alpha-androstanediol into the active prostatic, androgen dihydrotestosterone. Additionally, a nuclear transcription factor termed EPAS1, that is selectively expressed in endothelial cells and that activates endothelial cell-specific genes was isolated. We now propose to characterize the roles of these genes in the prostate and to isolate additional cDNAs encoding androgen metabolizing enzymes. In Specific Aim 1, cDNAs encoding androgen metabolizing enzymes will be isolated from prostate libraries by expression cloning in mammalian cells. The isolated cDNAs will be characterized with respect to DNA sequence, chromosomal location, tissue and cell type distribution, developmental expression patterns in the urogenital tract, and the biochemical parameters of the encoded enzyme. In Aim 2, the expression of these cDNAs, the oxidative 3alpha-hydroxysteroid dehydrogenase, and EPAS1, will be determined in prostate disease. Biochemical, immunochemical and molecular assays will be used to measure target protein and mRNA levels in BPH and prostatic cancer specimens provided by the Tissue Core Facility of this O?Brien Center. In Aim 3, the consequences of EPAS1 gene disruption in mice will be determined. As detailed in Preliminary Results, targeted gene disruption produced mice heterozygous for a null mutation at the Epas1 locus. Animals that are homozygous for the mutation will be characterized with respect to formation of the vasculature and angiogenesis. The Epas1 mutation will be crossed into nude mice and its effects on the propagation of prostatic tumor xenografts will be determined. Androgen metabolism and angiogenesis are crucial for the development and growth of the normal and pathologic prostate and we believe that the proposed studies will provide novel insight into these processes.